Clinical recurrence after therapy with cytotoxic agents presents a life threatening problem for a large number of cancer patients. It has been hypothesized that this form of therapy resistance is mediated by a small population of growth arrested cancer stem cells that survive treatment and initiate recurrence. Although intriguing, this hypothesis remains untested due to technical limitations that preclude direct detection of these growth arrested cancer cells within intact tumors. We have overcome this problem with the development of a dual fluorescent reporter system that permanently marks all cancer cells with the expression of a red fluorescent protein, while it distinguishes slow-cycling from rapidly proliferating cancer cells by their retention of a doxycycline repressible Histone 2B green fluorescent fusion protein (H2BGFP) in pulse-chase experiments. Using squamous cell carcinoma as a paradigm for hierarchically organized tumors, we are able to detect fast cycling and growth arrested cancer cells with tumor initiating potential. This application aims to test the hypothesis that: (1.) squamous cell carcinomas contain cancer stem cells that interconvert between fast and slow cycling states by responsive adaptation mechanisms; (2.) fast and slow cycling cancer stem cells are defined by characteristic molecular signatures that govern their proliferative behavior; and (3.) slow cycling cancer stem cells are inert to cytotoxic drugs and able to initiate recurrence after treatment. The results of our research are expected to positively impact the design of therapies and treatment of cancer patients as the identification of quiescent cancer stem cells and the mechanisms that govern their behavior presents a first step towards the identification of prognostic markers that predict which cancers will resist cytotoxic therapies It will also promote the development of novel combination therapies that specifically stimulate proliferation of quiescent cancer stem cells to increase their vulnerability to cytotoxic drugs, without affecting quiescence of normal, adult stem cells.